FIELD OF THE INVENTION
[0001] The invention relates to the fields of diagnostics and therapeutics, in particular
to a method of providing personalized management to brain cancer patients based on
the expression of certain genes in a sample from said patients, which certain serve
as treatment targets.
BACKGROUND OF THE INVENTION
Gliomas: Diagnosis and disease categorization.
[0002] A glioma is a type of cancer that starts in the brain or spine. It is called a glioma
because it arises from glial cells and/or its precursors. The most common site of
gliomas is the brain. Gliomas are classified by cell type, by grade, and by location.
Gliomas are named according to the specific type of cell they most closely resemble.
The main types of gliomas are:
- Ependymomas, gliomas derived from ependymal cells
- Astrocytomas, gliomas derived from astrocytes. The glioblastoma multiforme is the
most common astrocytoma.
- Oligodendrogliomas , gliomas derived from oligodendrocytes.
- Mixed gliomas, such as oligoastrocytomas, that contain cells from different types
of glia.
[0003] Gliomas are further categorized according to their grade, which is determined by
pathologic evaluation of the tumor. Thus we can distinguish between low-grade gliomas
that are well-differentiated (not anaplastic), are benign and portend a better prognosis
for the patient; and high-grade gliomas, that are undifferentiated or anaplastic,
are malignant and carry a worse prognosis.
[0004] Of numerous grading systems in use, the most common is the World Health Organization
(WHO) grading system for astrocytoma.
Treatment of brain gliomas.
[0005] The treatment for brain gliomas depends on the location, the cell type and the grade
of malignancy. Often, treatment is a combined approach, using surgery, radiation therapy,
and chemotherapy. The radiation therapy is in the form of external beam radiation
or the stereotactic approach using radiosurgery. Spinal cord tumors can be treated
by surgery and radiation. Temozolomide is a chemotherapeutic drug that is able to
cross the blood-brain barrier effectively and is being used in therapy. Despite these
approaches most high grade glioma patients succumb to their disease. New therapeutic
interventions to critical targets are needed to improve outcome in this patient population.
Glioblastoma multiforme
[0006] The glioblastoma multiforme (GBM, WHO grade IV) is a highly aggressive brain tumor
presenting as one of two subtypes with distinct clinical histories and molecular profiles.
The primary GBM presents acutely as a high-grade disease and the secondary GBM subtype
evolves from the slow progression of a low-grade disease.
[0007] Brown et al (J. of clinical Oncology., 2008, 5603-5609) describe a phase I/II trial of erlotinib combined with temozolomide in patients
suffering glioblastoma multiforme (GBM). These authors tried to correlate the response
of the patients with several molecular markers like EGFR, PTEN, P53 etc. but failed
to observe any correlation between survival and expression levels of said genes.
[0009] Van den Bent M. J. et al (JCO 2009;27:1268-1274) describes a recent randomized PHII EORTC trial. Patients with progressive GBM after
prior radiotherapy were randomly assigned to either erlotinib or a control arm that
received treatment with either temozolomide or carmustine (BCNU). The primary end
point was 6-month progression-free survival (PFS). Tumor specimens obtained at first
surgery were investigated for EGFR expression; EGFRvIII mutants; EGFR amplification;
EGFR mutations in exons 18, 19, and 21; and pAkt. No clear biomarker associated with
improved outcome to erlotinib was identified.
[0010] Smith J.S et al (J. National Cancer Institute 2001. 1246-1256) describe methods for predicting the survival of patients with anaplastic astrocytoma
and glioblastoma multiforme. These authors identify that mutation of PTEN and EGFR
amplification are independent prognostic markers for patients with anaplastic astrocytoma
and EGFR amplification is a survival marker for older patients with gliobastoma multiforme.
[0011] Prados M. D. et al (J Clin Oncol. 2009 27(4):579-84) describes a Ph II trial which evaluated erlotinib plus temozolomide during and after
radiation, patients treated with combination therapy (i.e. erlotinib and temozolomide)
had better survival. In addition, the study also evaluated several biomarkers and
found that methylation of the MGMT promoter along with PTEN expression was associated
with improved survival.
[0012] However, there is still a need for further markers and markers combinations useful
for predicting the clinical outcome of the glioma patients. A special area for diagnosis
and prognosis is the study of the biopsy sample. An integrated approach able to better
define patient outcome based on the most appropriate treatment using tumor profiles
will be critical, offering in addition to the glioma patient a better quality of life.
SUMMARY OF THE INVENTION
[0013] In a first aspect, the invention relates to a method for predicting the clinical
outcome of a subject suffering from glioma comprising determining the expression levels
of EGFR and/or polysomy and/or amplification of the EGFR locus on chromosome 7 and
the expression levels and/or the LOH levels of PTEN gene in a sample from the subject
and comparing said expression levels or the polysomy / amplification levels of EGFR
gene and the expression levels and/or the LOH levels of PTEN gene with standard reference
values, wherein low expression levels and/or high LOH levels of PTEN gene with respect
to said reference values and high expression levels and/or high polysomy / amplification
of EGFR gene are indicative of a good clinical outcome of the subject.
[0014] In a second aspect, the invention relates to a method for predicting the clinical
outcome of a subject suffering from glioma comprising determining the expression levels
and/or the LOH levels of PTEN gene in a sample from the subject and comparing said
expression levels and/or the LOH levels of PTEN gene with standard reference values,
wherein low expression levels and/or low LOH levels of PTEN gene with respect to said
reference values, is indicative of a poor clinical outcome of the subject.
[0015] In a third aspect, the invention relates to a Kit comprising agents capable of specifically
detecting the expression levels and/or the polysomy of EGFR and the expression levels
and/or the LOH of PTEN genes.
BRIEF DESCRIPTION OF THE FIGURES
[0016]
Figure 1. Kaplan-Meier survival curve illustrating time from first surgery to death
/ end-of follow-up of only (Pure) GBM patients, astrocyoma patients and oligodendroglioma
patients.
Figure 2. Kaplan-Meier curve estimating survival from first surgery to death / recurrence
/ progression or end-of-follow-up of only (Pure) GBM patients, astrocyoma patients
and oligodendroglioma patients.
Figure 3. Kaplan-Meier survival curve demonstrating stratification of patients in
the glioblastoma (GBM) /anaplastic astrocytoma and mixed group with PTEN LOH (p=0.04).
Figure 4. Kaplan-Meier survival curve demonstrating stratification of patients in
the glioblastoma (GBM) /anaplastic astrocytoma and mixed group as having both EGFR
AMP/HP (amplification - high polysomy) and PTEN LOH (p=0.034).
Figure 5. Representative images of glioblastoma multiforme specimens stained with
H&E (Hematoxylin and Eosin) (A). Example of FISH experiments where AMP/HP EGFR DNA
FISH (B) and monosomy PTEN (C) are illustrated.
DETAILED DESCRIPTION OF THE INVENTION
[0017] In order to facilitate the understanding of the invention described in this patent
application, the meaning of some terms and expressions in the context of the invention
are explained below:
The term "subject" refers to a member of a mammal animal species, and includes, but
is not limited thereto, domestic animals, primates and humans; the subject is preferably
a human being, male or female, of any age or race. Alternatively, the term "individual"
is also sometimes used in this description to refer to human beings.
The term "protein" refers to a molecular chain of amino acids, linked by covalent
or non-covalent bonds. The term includes all the forms of post-translational modifications,
for example, glycosylation, phosphorylation or acetylation.
The term "antibody" refers to a protein with the capacity to specifically bind to
an "antigen". The term "antibody" comprises recombinant antibodies, monoclonal antibodies,
or polyclonal antibodies, intact, or fragments thereof which maintain the capacity
to bind to the antigen, combibodies, etc., both human or humanised and of non-human
origin.
The term "primer oligonucleotide", as used in the present invention, refers to a nucleotide
sequence, which is complementary to a nucleotide sequence of a selected gene. Each
primer oligonucleotide hybridises with its target nucleotide sequence and acts as
an initiation point for DNA polymerisation.
[0018] The authors of the present invention have found that the clinical outcome of patients
suffering from glioma cancer correlates with levels and/or the polysomy / amplification
levels of EGFR gene and the expression levels and/or the LOH levels of PTEN.
[0019] Thus, in a first aspect, the invention relates to a method for predicting the clinical
outcome of a subject suffering from glioma that comprises
- a) determining the expression levels and/or the polysomy/ amplification levels of
EGFR gene and the expression levels and/or the LOH levels of PTEN gene in a sample
from the subject and
- b) comparing said expression levels or the polysomy / amplification levels of EGFR
gene and the expression levels and/or the LOH levels of PTEN gene with standard reference
values,
wherein low expression levels and/or high LOH levels of PTEN gene with respect to
said standard reference values and high expression levels and/or high polysomy of
EGFR gene with respect to said standard reference values are indicative of a good
clinical outcome of the subject. In the present invention a "glioma" is a type of
cancer that starts in the brain or spine. It is called a glioma because it arises
from glial cells. The most common site of gliomas is the brain. Gliomas are classified
by cell type, by grade, and by location.
[0020] Gliomas are named according to the specific type of cell they most closely resemble.
The main types of gliomas are:
- Ependymomas, gliomas derived from ependymal cells
- Astrocytomas, gliomas derived from astrocytes. Astrocytoma are derived in pilocytic,
diffuse, anaplastic, and glioblastoma multiforme. The glioblastoma multiforme is the
most common astrocytoma.
- Oligodendrogliomas , gliomas derived from oligodendrocytes.
- Mixed gliomas, such as oligoastrocytomas, that contain cells from different types
of glia.
[0021] In a preferred embodiment, the glioma is a gliobastoma multiforme (GBM) and more
preferably the gliobastoma is an early gliobastoma.
[0022] The gliobastoma multiforme is the most common and malignant form of glial tumors
and is composed of a heterogenous mixture of poorly differentiated malignant astrocytes
and dysplastic endothelial cells. It primarily effects adults, involves the cerebral
hemispheres and has a rapid disease course which often leads to death.
[0023] In the present invention "clinical outcome" is understood as the expected course
of a disease. It denotes the doctor's prediction of how a subject's disease will progress,
and whether there is chance of recovery or recurrence.
[0024] The prediction of the clinical outcome can be done by using any endpoint measurements
used in oncology and known to the skilled practitioner. Useful endpoint parameters
to describe the evolution of a disease include:
- disease-free progression which, as used herein, describes the proportion of patients
in complete remission who have had no recurrence of disease during the time period
under study.
- objective response, which, as used in the present invention, describes the proportion
of treated people in whom a complete or partial response is observed.
- tumor control, which, as used in the present invention, relates to the proportion
of treated people in whom complete response, partial response, minor response or stable
disease ≥ months is observed.
- progression free survival which, as used herein, is defined as the time from start
of treatment to the first measurement of cancer growth.
- six-month progression free survival or PFS6" rate which, as used herein, relates to
the percentage of people wherein free of progression in the first six months after
the initiation of the therapy and
- median survival which, as used herein, relates to the time at which half of the patients
enrolled in the study are still alive.
[0025] A good clinical outcome is understood as a situation where at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% of the patients have a positive result regarding the
endpoint parameters described above.
[0026] The term "sample" as used herein, relates to any sample which can be obtained from
the patient. The present method can be applied to any type of biological sample from
a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva, semen, sputum,
cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts and the like).
In a particular embodiment, said sample is a tumour tissue sample or portion thereof
In a more particular embodiment, said tumor tissue sample is a brain tumor tissue
sample from a patient suffering from brain cancer. Said sample can be obtained by
conventional methods, e.g., biopsy, by using methods well known to those of ordinary
skill in the related medical arts. Methods for obtaining the sample from the biopsy
include gross apportioning of a mass, or microdissection or other art-known cell-separation
methods. Tumour cells can additionally be obtained from fine needle aspiration cytology.
In order to simplify conservation and handling of the samples, these can be formalin-fixed
and paraffin-embedded or first frozen and then embedded in a cryosolidifiable medium,
such as OCT-Compound, through immersion in a highly cryogenic medium that allows for
rapid freeze.
[0027] The samples may be obtained from subjects previously diagnosed with glioma (patients),
or from subjects who have not been previously diagnosed with glioma, or from patients
diagnosed with glioma who are undergoing treatment, or from subjects diagnosed with
glioma who have been previously treated.
[0028] In a preferred embodiment, the sample is a tumor sample that contains a substantially
number of tumor cells.
[0029] PTEN is the Phosphatase and tensin homolog protein also known as BZS; MHAM; TEP1;
MMAC1; PTEN1; 10q23del or MGC11227. The PTEN is a protein (which in humans is encoded
by the PTEN gene (RefSEq ID NM_000314 SEQ ID NO: 1, Protein reference NP_000305.3
SEQ ID NO: 2) (
Steck PA, et al. 1997 Nat. Genet. 15 (4): 356-62 2). PTEN acts as a tumor suppressor gene through the action of its phosphatase protein
product. This phosphatase is involved in the regulation of the cell cycle, preventing
cells from growing and dividing too rapidly. Mutations of this gene contribute to
the development of certain cancers (
Chu EC, et al. 2004 Med. Sci. Monit. 10 (10): RA235-41 3). It does exist homologs in other species (mice (NM_008960.2, SEQ ID NO: 3), rat
(NM 031606.1, SEQ ID NO: 4) and dog (NM 001003192, SEQ ID NO: 5), etc).
[0030] The protein encoded this gene is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase.
It contains a tensin like domain as well as a catalytic domain similar to that of
the dual specificity protein tyrosine phosphatases. Unlike most of the protein tyrosine
phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates.
It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate
in cells and functions as a tumor suppressor by negatively regulating Akt/PKB signaling
pathway (
Hamada K, et al 2005 Genes Dev 19 (17): 2054-65)
[0031] The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the cell-surface
receptor for members of the epidermal growth factor family (EGF-family) of extracellular
protein ligands. (
Herbst RS (2004). Int. J. Radiat. Oncol. Biol. Phys. 59 (2 Suppl): 21-6. 1) The epidermal growth factor receptor is a member of the ErbB family of receptors.
The EGFR is a protein which in humans is encoded by different isoforms: EGFR transcript
variant 1(NM_005228.3, SEQ ID NO: 6), transcript variant 2 (NM_201282.1, SEQ ID NO:
7), transcript variant 3 (NM 201283.1, SEQ ID NO: 8) and transcript variant 4 (NM_201284.1,
SEQ ID NO: 9). It does exist homologes in other species (mice (NM_207655.2, SEQ ID
NO: 10 and NM 007912.4, SEQ ID NO: 11), rat (NM_031507.1, SEQ ID NO: 12) and dog (XM_533073.2,
SEQ ID NO: 13), etc)
[0032] The method of the invention requires determining the expression levels of the EGFR
gene and PTEN gene. As the person skilled in the art understands, the expression levels
of the PTEN gene, and EGFR gene can be determined by measuring the levels of mRNA
encoded by said genes, measuring both the levels of proteins encoded by said genes,
i.e. EGFR protein, and PTEN protein, and the levels of variants thereof and through
the use of surrogates (DNA copy number) for associating gene level with mRNA and protein
product of said gene.
[0033] Variants of the EGFR protein, and PTEN protein may be (i) one in which one or more
of the amino acid residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such substituted amino
acid residue may or may not be one encoded by the genetic code, (ii) one in which
there are one or more modified amino acid residues, e.g., residues that are modified
by the attachment of substituent groups, (iii) one in which the protein is an alternative
splice variant of the proteins of the present invention and/or (iv) fragments of the
proteins. The fragments include proteins generated via proteolytic cleavage (including
multi-site proteolysis) of an original sequence. Variants are deemed to be within
the scope of those skilled in the art from the teaching herein.
[0034] As known in the art the "similarity" between two proteins is determined by comparing
the amino acid sequence and its conserved amino acid substitutes of one protein to
a sequence of a second protein. Variants according to the present invention includes
amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%,
or 95% similar or identical to the original amino acid sequence. The degree of identity
between two proteins is determined using computer algorithms and methods that are
widely known for the persons skilled in the art. The identity between two amino acid
sequences is preferably determined by using the BLASTP algorithm [BLASTManual, Altschul,
S., et al., NCBI NLM NIH Bethesda, Md. 20894,
Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].
[0035] The proteins can be post-translationally modified. For example, post-translational
modifications that fall within the scope of the present invention include signal peptide
cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation,
protein folding and proteolytic processing, etc. Additionally, the proteins may include
unnatural amino acids formed by post-translational modification or by introducing
unnatural amino acids during translation.
[0036] In a preferred embodiment, the determination of the expression levels of the EGFR
gene and PTEN gene can be carried out by measuring the expression levels of the mRNA
encoded by the EGFR gene and PTEN gene. For this purpose, the biological sample may
be treated to physically or mechanically disrupt tissue or cell structure, to release
intracellular components into an aqueous or organic solution to prepare nucleic acids
for further analysis. The nucleic acids are extracted from the sample by procedures
known to the skilled person and commercially available. RNA is then extracted from
frozen or fresh samples by any of the methods typical in the art, for example,
Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.),
Cold Spring Harbor Laboratory Press, New York, (1989). Preferably, care is taken to avoid degradation of the RNA during the extraction
process.
[0037] In a particular embodiment, the expression level is determined using mRNA obtained
from a formalin-fixed, paraffin-embedded tissue sample. mRNA may be isolated from
an archival pathological sample or biopsy sample which is first deparaffinized. An
exemplary deparaffinization method involves washing the paraffinized sample with an
organic solvent, such as xylene, for example. Deparaffinized samples can be rehydrated
with an aqueous solution of a lower alcohol. Suitable lower alcohols, for example
include, methanol, ethanol, propanols, and butanols. Deparaffinized samples may be
rehydrated with successive washes with lower alcoholic solutions of decreasing concentration,
for example. Alternatively, the sample is simultaneously deparaffinized and rehydrated.
The sample is then lysed and RNA is extracted from the sample.
[0038] While all techniques of gene expression profiling (RT-PCR, SAGE, or TaqMan) are suitable
for use in performing the foregoing aspects of the invention, the gene mRNA expression
levels are often determined by reverse transcription polymerase chain reaction (RT-PCR).
The detection can be carried out in individual samples or in tissue micro arrays.
[0039] In order to normalize the values of mRNA expression among the different samples,
it is possible to compare the expression levels of the mRNA of interest in the test
samples with the expression of a control RNA. A "Control RNA" as used herein, relates
to a RNA whose expression levels do not change or change only in limited amounts in
tumor cells with respect to non-tumorigenic cells. Preferably, the control RNA are
mRNA derived from housekeeping genes and which code for proteins which are constitutively
expressed and carry out essential cellular functions. Preferred housekeeping genes
for use in the present invention include β-2-microglobulin, ubiquitin, 18-S ribosomal
protein, cyclophilin, GAPDH and actin. In a preferred embodiment, the control RNA
is β-actin mRNA. In one embodiment relative gene expression quantification is calculated
according to the comparative Ct method using β-actin as an endogenous control and
commercial RNA controls as calibrators. Final results, are determined according to
the formula 2-(ΔCt sample-ΔCt calibrator), where ΔCT values of the calibrator and
sample are determined by subtracting the CT value of the target gene from the value
of the β-actin gene.
[0040] The determination of the level of expression of the EGFR gene and PTEN gene needs
to be correlated with the standard reference values. Standard reference values which
correspond to the median value of expression levels of EGFR gene and PTEN gene measured
in a collection of samples from healthy patients. Once this median value is established,
the level of this marker expressed in tumor tissues from patients can be compared
with this median value, and thus be assigned a level of "low," "normal" or "high".
The collection of samples from which the reference level is derived will preferably
be constituted from healthy persons from the same age as the patients. In any case
it can contain a different number of samples. In a more preferred embodiment, the
samples are biopsy brain samples. Preferably the collection should be sufficient to
provide an accurate reference level. Preferably the number of samples used for make
the reference values is more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200,
500 samples.
[0041] In a particular embodiment, an increase in expression above the reference value of
at least 1.1-folk, 1.5-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,
60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more compared with the reference
value is considered as "high" expression. In a particular embodiment, a decrease in
expression below the reference value of at least 0.9-fold, 0.75-fold, 0.2-fold, 0.1-fold,
0.05-fold, 0.025-fold, 0.02-fold, 0.01-fold, 0.005-fold or even less compared with
the reference value is considered as "low" expression.
[0042] Alternatively, in another particular embodiment, the expression levels of the EGFR
gene, and/or PTEN gene can be determined by measuring both the levels of proteins
encoded by said genes, i.e. EGFR protein, and PTEN protein, and the levels of variants
thereof.
[0043] The determination of the expression levels of the PTEN or EGFR proteins and the variants
thereof can be carried out by immunological techniques such as e.g. ELISA, Western
Blot or immunofluorescence. Western blot is based on the detection of proteins previously
resolved by gel electrophoreses under denaturing conditions and immobilized on a membrane,
generally nitrocellulose by the incubation with an antibody specific and a developing
system (e.g. chemoluminiscent). The analysis by immunofluorescence requires the use
of an antibody specific for the target protein for the analysis of the expression
and subcellular localization by microscopy. Generally, the cells under study are previously
fixed with paraformaldehyde and permeabilised with a non-ionic detergent. ELISA is
based on the use of antigens or antibodies labelled with enzymes so that the conjugates
formed between the target antigen and the labelled antibody results in the formation
of enzymatically-active complexes. Since one of the components (the antigen or the
labelled antibody) are immobilised on a support, the antibody-antigen complexes are
immobilised on the support and thus, it can be detected by the addition of a substrate
which is converted by the enzyme to a product which is detectable by, e.g. spectrophotometry
or fluorometry. This technique does not allow the exact localisation of the target
protein or the determination of its molecular weight but allows a very specific and
highly sensitive detection of the target protein in a variety of biological samples
(serum, plasma, tissue homogenates, postnuclear supernatants, ascites and the like).
In a preferred embodiment, the EGFR and PTEN proteins are detected by immunohistochemistry
(IHC) analysis using thin sections of the biological sample immobilised on coated
slides. The sections are then deparaffinised, if derived from a paraffinised tissue
sample, and treated so as to retrieve the antigen. The detection can be carried out
in individual samples or in tissue microarrays. In another embodiment the method used
is a proteomic array
[0044] Any antibody or reagent known to bind with high affinity to the target protein can
be used for detecting the amount of target protein. It is preferred nevertheless the
use of antibody, for example polyclonal sera, hybridoma supernatants or monoclonal
antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies,
tetrabodies and humanised antibodies.
[0045] In yet another embodiment, the determination of EGFR and PTEN proteins expression
levels can be carried out by constructing a tissue microarray (TMA) containing the
patient samples assembled, and determining the expression levels of EGFR and PTEN
proteins by immunohistochemistry techniques. Immunostaining intensity can be evaluated
by two different pathologists and scored using uniform and clear cut-off criteria,
in order to maintain the reproducibility of the method. Discrepancies can be resolved
by simultaneous re-evaluation. Briefly, the result of immunostaining can be recorded
as negative expression (0) versus positive expression, and low expression (1+) versus
moderate (2+) and high (3+) expression, taking into account the expression in tumoral
cells and the specific cut-off for each marker. As a general criterion, the cutoffs
were selected in order to facilitate reproducibility, and when possible, to translate
biological events.
[0046] The determination of the level of expression of the EGFR gene and PTEN gene needs
to be correlated with the reference values which correspond to the median value of
expression levels of EGFR gene and PTEN gene measured in a collection of brain tissue
samples from healthy patients. Preferably the collection should be sufficient to provide
an accurate reference level. Preferably the number of samples used for make the reference
values is more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500 samples.
[0047] In a preferred embodiment the sample is a biopsy. Once this median value is established,
the level of this marker expressed in tumor tissues from patients can be compared
with this median value, and thus be assigned a level of "low," "normal" or "high."
[0048] Polysomy and/or amplification of the EGFR locus on chromosome 7 in the present invention
is understood as the presence of more than one copy of the EGFR locus on the chromosome
7.
[0049] In the present invention the term "LOH" refers to loss of heterozygosity. LOH in
a cell represents the loss of normal function of one allele of a gene in which the
other allele was already inactivated. This term is mostly used in the context of oncogenesis;
after an inactivating mutation in one allele of a tumor suppressor gene occurs in
the parent's germline cell, it is passed on to the zygote resulting in an offspring
that is heterozygous for that allele. In oncology, loss of heterozygosity occurs when
the remaining functional allele in a somatic cell of the offspring becomes inactivated
by mutation. In the case of PTEN LOH, this results in no normal tumor suppressor being
produced.
[0050] In a particular embodiment, the determination of the polysomy / amplification of
EGFR and the LOH levels of PTEN can be measured in the DNA obtained from the tumor
cells according to standard procedures such as quantitative PCR or comparative genomic
hybridization to microarray technologies; or in the tumor cells from the paraffined-embedded
section or from the cytology preparation by FISH using appropriate molecular probes.
In a particular embodiment, the detection of the polysomy / amplification of EGFR
and the LOH levels of PTEN is carried out by a hybridization-based assay. In a particular
embodiment, the detecting step of the method of the invention comprises contacting
the nucleic acid sample with one or more nucleic acid probes each of which selectively
binds to a target polynucleotide sequence on the chromosome region of EGFR or PTEN,
under conditions in which the probe forms a stable hybridization complex with the
target polynucleotide sequence; and detecting the hybridization complex. In a particular
embodiment, the nucleic acid probes used in the method of the present invention are
labelled with a fluorophore.
[0051] In a particular embodiment, the step of detecting the hybridization complex comprises
determining the copy number of the target polynucleotide sequence, thereby determining
the presence of the polysomy or the LOH.
[0052] In a preferred embodiment of the invention, said hybridization-based assay is selected
from the group consisting of Southern blot, PCR, in situ hybridization (ISH) fluorescence
ISH (FISH) and comparative genomic hybridization (CGH). In a more preferred embodiment,
the method is a comparative genomic hybridization assay.
[0053] In a particular embodiment of the invention, said hybridization-based assay is an
array-based assay. In a particular embodiment, once the sample has been obtained and
the total DNA has been extracted, genome-wide analysis of DNA copy number changes
by comparative genomic hybridization (CGH) is carried out. In general, for a typical
CGH measurement, total genomic DNA is isolated from test and reference cell populations,
differentially labeled and hybridized to a representation of the genome that allows
the binding of sequences at different genomic locations to be distinguished. Hybridization
reactions can be performed under conditions of different stringency. The stringency
of a hybridization reaction includes the difficulty with which any two nucleic acid
molecules will hybridize to one another. Preferably, each hybridizing polynucleotide
hybridizes to its corresponding polynucleotide under reduced stringency conditions,
more preferably stringent conditions, and most preferably highly stringent conditions.
[0054] The amount of specimen DNA is frequently a constraint on CGH measurements. Typical
array CGH procedures use 300 ng to 3 µg of specimen DNA in the labelling reaction,
equivalent to approximately 50.000 to 500.000 mammalian cells. Usually, random primer
labeling protocols are employed, which also amplifies the DNA, so that several micrograms
are used in the hybridization.
[0055] Array CGH has been implemented using a wide variety of techniques. In a particular
embodiment, array CGH is carried out using arrays from large-insert genomic clones
such as bacterial artificial chromosomes (BACs). The general principles and conditions
for detection of nucleic acids, such as using array CGH (comparative genomic hybridization
(CGH) to BAC microarrays), are well known for the skilled person in the art. This
technique allows scanning the entire genome for DNA copy number changes therefore
allowing quantitative detection of DNA copy number variation in tumor genomes with
high resolution (
Pinkel D, et al. Nat Genet 1998; 20(2):207-11 and
Hodgson G, et al. Nat Genet 2001;29(4):459-64).
[0056] As an illustrative non limitative example, in the array CGH carried out by the method
of the present invention test tumor and reference genomic DNAs can be labeled by random
priming using Cy3 and Cy5 fluorophores. Then, the images of the arrays may be analysed
using, for example, a cooled coupled device (CCD) camera and appropriate software.
[0057] The major technical challenge of array CGH is generating hybridization signals that
are sufficiently intense and specific so that copy number changes can be detected.
The signal intensity on an array element is affected by a number of factors including
the base composition, the proportion of repetitive sequence content, and the amount
of DNA in the array element available for hybridization.
[0058] Array elements made from genomic BAC clones typically provide more intense signals
than elements employing shorter sequences such as cDNAs, PCR products, and oligonucleotides.
The higher signals from the more complex array elements result in better measurement
precision, allowing detection of single-copy transition boundaries-even in specimens
with a high proportion of normal cells.
[0059] In another preferred embodiment of the invention the method used is FISH (fluorescence
in situ hybridization) or FISH plus spectral karotype (SKY) (Liehr T. et al 2008 Fluorescence
In Situ Hybridization (FISH) - Application Guide, Springer Berlin Heidelberg)
[0060] FISH is a cytogenetic technique used to detect and localize the presence or absence
of specific DNA sequences on chromosomes. FISH uses fluorescent probes that bind to
only those parts of the chromosome with which they show a high degree of sequence
similarity. A s
"probe" is understood any ribopolynucleotide or desoxiribopolynucleotide sequence that specifically
binds to only those parts of the chromosome with which they show a high degree of
sequence similarity. The probe must be large enough to hybridize specifically with
its target but not so large as to impede the hybridization process.
[0061] It does exist many different FISH probes that can be used in the present invention
like without limitation, bacterial artificial chromosomes (BACs), Tiling Oligonucleotide
Probes (TOPs), etc. The design of FISH probes is well know for a person skilled in
the art (
Bayani J, Squire JA..Curr Protoc Cell Biol. 2004 Sep; Chapter 22:Unit 22.4;
Bayani J, Squire J.Curr Protoc Cell Biol. 2004 Oct;Chapter 22:Unit 22.5.;
Navin, N. et al. Bioinformatics, Volume 22, Number 19, 1 October 2006 , pp. 2437-2438(2))
Publisher: Oxford University Press)
[0062] The probe can be tagged directly with fluorophores, with targets for antibodies or
with biotin. Tagging can be done in various ways, such as nick translation, or PCR
using tagged nucleotides.
[0063] The sample can be fixed and in paraffin embedded, thus an additionally step of deparafination
may be performed.
[0064] For hybridization, an interphase or metaphase chromosome preparation is produced.
The chromosomes are firmly attached to a substrate, usually glass. Repetitive DNA
sequences must be blocked by adding short fragments of DNA to the sample. The probe
is then applied to the chromosome DNA and incubated for approximately 12 hours while
hybridizing. Several wash steps remove all unhybridized or partially-hybridized probes.
After standard post hybridization washes the slides are stained with the DNA staining
probe such DAPI and mounted with a mounting agent such as antifade.
[0065] The results are then visualized and quantified using a microscope that is capable
of exciting the dye and recording images. If the fluorescent signal is weak, amplification
of the signal may be necessary in order to exceed the detection threshold of the microscope.
Fluorescent signal strength depends on many factors such as probe labeling efficiency,
the type of probe, and the type of dye. Fluorescently-tagged antibodies or streptavidin
are bound to the dye molecule. These secondary components are selected so that they
have a strong signal. In a preferred embodiment, prior to imaging all slides are evaluated
by a pathologist and regions of interest are identified based on histopathologic and
quality criteria including, without excluding others, tumor content, appropriate fixation,
necrosis and vascularity.
[0066] FISH experiments designed to detect or localize gene expression within cells and
tissues rely on the use of a reporter gene, such as one expressing green fluorescent
protein, to provide the fluorescence signal.
[0067] In an alternative technique to interphase or metaphase preparations, fiber FISH,
interphase chromosomes are attached to a slide in such a way that they are stretched
out in a straight line, rather than being tightly coiled, as in conventional FISH,
or adopting a random conformation, as in interphase FISH. This is accomplished by
applying mechanical shear along the length of the slide, either to cells that have
been fixed to the slide and then lysed, or to a solution of purified DNA. The extended
conformation of the chromosomes allows dramatically higher resolution - even down
to a few kilobases.
[0068] In a preferred embodiment, parallel to the detection of the polysomy / amplification
of EGFR locus and LOH of PTEN, FISH control probes for chromosomes 10 and 7 are used.
Those "FISH control probes" are probes that binds specific for the individual chromosomes,
thus allowing the determination of the chromosome number. In a preferred embodiment,
the control probes are directed to alpha satellite sequences. Alpha satellite sequences,
whilst highly repetitive, are specific to each individual chromosome. These sequences
flank the centromeres and can present a target measured in megabases. In a preferred
embodiment, these FISH control probes would be label with different colors than the
EGFR and PTEN probes.
[0069] In a preferred embodiment, the PTEN FISH probe is a probe that hybridizes to the
10q23 region on chromosome 10 and contains sequences that flank both the 5' and 3'
ends of the PTEN gene; in a more preferred embodiment the probe has between 300 and
400 kb. In a more preferred embodiment, the FISH probe for PTEN and the control probe
for chromosome 10 are the LSI PTEN (10q23) / CEP 10 dual color probe for PTEN (Vysis,
Abbot Molecular) (
Goberdhan,D., et al.Human Molecular Genetics 12 (2) (2003): 239-248.
Eng, C., et al.Human Mutation 22 (2003): 183-198.
Sasaki, H., et al. Am. J. of Pathology 159 (1) (2001): 359-367). Other probes are described in
Cairns et al. (1997. Cancer Res 57; 4997-5000) and
Hermans et al, (2004 Genes Chrom Cancer, 39; 171-184). The LSIS PTEN (10q23) is labeled with SpectrumOrange. The CEP 10 SpectrumGreen
probe hybridizes to alpha satellite sequences specific to chromosome 10.
[0070] The EGFR FISH probe is a probe that hybridizes to the 7p12 region of the chromosome
7 and that contains the entire EGFR gene. In a more preferred embodiment, the FISH
probe for PTEN and the control probe for chromosome 10 are the LSI EGFR/CEP 7 dual
color probe (Vysis, Abbot Molecular)
[0071] In a particular embodiment the EGFR Probe is labeled in SpectrumOrange and covers
an approximately 300 kb region of the 7p12 (
Bredel, M., et al. (1999) Clin Cancer Res 5, 1786-92.
Harris, A., et al. (1989) J Steroid Biochem 34, 123-31.
Kitagawa, Y., et al. (1996) Clin Cancer Res 2, 909-14.
Neal, D.E., et al. (1990) Cancer 65, 1619-25.
Osaki, A., et al. (1992) Am J of Surg 164, 323-6.
Pavelic, K., et al. (1993) Anticancer Res 13, 1133-7.
Sauter, G., et al. (1996) Am J Pathol 148, 1047-53.
Torregrosa, D., et al. (1997) Clin Chim Acta 262, 99-119). The CEP 7 probe, labeled in SpectrumGreen, hybridizes to the alpha satellite DNA
located at the centromere of chromosome 7 (7p11.1-q11.1).
[0074] The determination of the level of the polysomy / amplification of EGFR and the level
of LOH of PTEN, needs to be correlated with a reference value. Said reference values
are generated by the person skill in the art in form of a table that divide the patient
in ascending number of copies of the EGFR gene or regarding to the LOH degree and
ascending number of copies of the TEN gene.
[0075] For EGFR, the reference values can be generated, without limitation, using for example,
the classification of
Cappuzzo et al. (JNCI 2005;4:643-55). In this classification, the patients are classified into six strata with ascending
number of copies of the EGFR gene per cell according to the frequency of tumor cells
with specific number of copies of the EGFR gene per chromosome 7. As it was mentioned
before, control probes that detect the chromosome 7 for obtaining a ratio number of
copies of EGFRgene/chromosome 7 are commercially available. For example, the CEP 7
SpectrumGreen probe (Vysis) that hybridizes to alpha satellite sequences specific
to chromosome 7, can be used.
[0076] An non limitative example of a table for classifying a patient attending to the polysomy
of EGFR is:
- 1) disomy (D): ≤ copies in 90% of the cells of the sample;
- 2) low trisomy (LT): ≤ 2 copies in ≥ 40% of the cells of the sample or 3 copies in
10% - 40% of the cells of the sample or ≥ 4 copies in <10% of the cells of the sample;
- 3) high trisomy (HT): ≤ 2 copies in ≥ 40% of the cells of the sample or 3 copies in
40% of the cells of the sample or ≥ 4 copies in <10% of the cells of the sample;
- 4) low polysomy (LP): ≥ 4 copies in 10% - 40% of the cells of the sample;
- 5) high polysomy (HP): ≥ 4 copies in ≥ 40% of the cells of the sample or the presence
of amplification (presence of tight EGFR gene clusters and a ratio of EGFR gene to
chromosome of ≥ 2 or ≥ 15 copies of EGFR per cell in ≥ 10% of analyzed cells of the
sample).
[0077] As a person skilled in the art would understand, the method of the invention can
be made using more than one sample of a patient. In such a case, it is consider that
exist "high polysomy levels of EGFR" when more than 50%, more than 60%, more than
70%, more than 80%, more than 90% of the samples of the patient are classified as
"high polysomy" according to the classification criteria described before. Similarly,
it is consider that exist "high LOH levels of PTEN" when more than 50%, more than
60%, more than 70%, more than 80%, more than 90% of the samples of the patient are
classified as "LOH" according to the classification criteria described before.
[0078] For the generation of a table for the clarification of the patients attending to
the LOH and number of copies of the PTEN gene, the person skilled in the art would,
for example clarified the patients into 4 strata with ascending number of copies of
the PTEN gene per cell according to the frequency of tumor cells with specific number
of copies of the PTEN gene per chromosome 10. As it was mentioned before, control
probes that detect the chromosome 10 for obtaining a ratio number of copies of PTEN
gene/chromosome 10 are commercially available. For example the CEP 10 SpectrumGreen
probe (Vysis) that hybridizes to alpha satellite sequences specific to chromosome
10, can be used.
[0079] An non limitative example of a table for classifying a patient attending to the polysomy/LOH
degrees is:
- 1) Disomy (D): 2 copies of each probe in 90% of cells
- 2) LOH = <2 copies of PTEN probe in 10% of cells (includes cromosome 10 monosomy or
disomy with PTEN loss, always in >10% cells)
- 3) Polysomy (P) = ≥ 3 copies each probe (PTEN+ CEP 10) in >10% of cells. It does not
discriminate between high and low polisomy, or chromosome 10 trisomy)
- 4) Amplified (AMP) = defined by a ratio of PTEN gene to chromosome 10 of ≥ 2 er cell
in ≥ 10% of analyzed cells.
[0080] In a last step of the method, low expression levels and/or high LOH levels of PTEN
gene with respect to said reference values and high expression levels and/or high
polysomy / amplification of EGFR gene are indicative of a good clinical outcome of
the subject.
[0081] As a person skilled in the art understand, the samples can also be considered not
appropriated for be include in any of the classification strata. Thus, in a preferred
embodiment, additionally, samples sections of tissue from the patient are stained
with colorants as hematoxylin and eosin (H&E) and reviewed by two or more persons
skilled in the art (i.e pathologists) to assess overall tumor content, necrosis and
overall quality (e.g. thermal cautery effect, fixation with morphology etc). In a
preferred embodiment the samples used for determining the expression levels and/or
the polysomy levels of EGFR gene and the expression levels and/or the LOH levels of
PTEN gene, have at least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% of tumor tissue in the sample, and have an
appropriate fixation with acceptable morphology.
[0082] The findings of the inventors allow the determination of the clinical outcome of
a patient suffering glioma measuring the expression levels and/or the LOH levels of
PTEN gene. Thus, in a second aspect, the invention relates to a method for predicting
the clinical outcome of a subject suffering from glioma comprising
- (a) determining the expression levels and/or the LOH levels of PTEN gene in a sample
from the subject and
- (b) comparing said expression levels and/or the LOH levels of PTEN gene with standard
reference values,
wherein low expression levels and/or high LOH levels of PTEN gene with respect to
said reference values, is indicative of a poor clinical outcome of the subject.
[0083] The term glioma has been previously defined.
[0084] In a preferred embodiment, the glioma is a gliobastoma multiforme (GMB) and more
preferably the gliobastoma is an early gliobastoma.
[0085] In a preferred embodiment, the clinical outcome is measured as survival.
[0086] The term "sample" has been previously defined and can be applied to any type of biological
sample from a patient, such as a biopsy sample, tissue, cell or fluid (serum, saliva,
semen, sputum, cerebral spinal fluid (CSF), tears, mucus, sweat, milk, brain extracts
and the like). In a particular embodiment, said sample is a tumour tissue sample or
portion thereof In a more particular embodiment, said tumor tissue sample is a brain
tumor tissue sample from a patient suffering from glioma or a formalin embedded brain
tissue sample.
[0087] The methods for determining the expression levels and the LOH of PTEN have been described
above as well as the standard reference values used. In a preferred embodiment, the
expression levels of PTEN gene are measured by determining mRNA and/or protein expression
level of said gene.
[0088] In a preferred embodiment, the polysomy levels and the LOH levels are determined
by FISH.
[0089] In another aspect, the invention relates to a kit useful in the implementation of
the methodology described herein.
[0090] Thus, in another aspect, the invention relates to a kit comprising a set of agents
capable of specifically detect the expression levels and/or the polysomy / amplification
of EGFR and the expression levels and/or the LOH of PTEN genes and, optionally, a
reagent for detecting a housekeeping gene or the protein encoded by said housekeeping
gene and/or a reagent for detecting the chromosomes 7 and 10.
[0091] In a particular embodiment of the kit of the invention, the agents of the kit are
capable of specifically detecting the mRNA levels of EGFR and/or PTEN genes or the
levels of EGFR and/or PTEN proteins.
[0092] Nucleic acids capable of specifically hybridizing with the EGFR and/or PTEN genes
can be one or more pairs of primer oligonucleotides for the specific amplification
of fragments of the mRNAs (or of their corresponding cDNAs) of said genes and/or one
or more probes for the identification of one or more genes selected from said genes.
Nucleic acids capable of specifically hybridizing with the EGFR and/or PTEN genes
can be as well FISH probes.
[0093] Antibodies, or a fragment thereof, capable of detecting an antigen, capable of specifically
binding to EGFR and/or PTEN proteins or to variants thereof are, for example, monoclonal
and polyclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies,
triabodies, tetrabodies and humanised antibodies.
[0094] Said reagents, specifically, the probes and the antibodies, may be fixed onto a solid
support, such as a membrane, a plastic or a glass, optionally treated in order to
facilitate fixation of said probes or antibodies onto the support. Said solid support,
which comprises, at least, a set of antibodies capable of specifically binding to
EGFR and/or PTEN proteins or to variants thereof, and/or probes specifically hybridized
with the EGFR and/or PTEN genes, may be used for the detection of the expression levels
by means of array technology.
[0095] The kits of the invention optionally comprise additional reagents for detecting a
polypeptide encoded by a housekeeping gene or the mRNA encoded by said housekeeping
genes. The availability of said additional reagent allows the normalization of measurements
taken in different samples (e.g. the test sample and the control sample) to exclude
that the differences in expression of the different biomarkers are due to a different
amount of total protein in the sample rather than to real differences in relative
expression levels. Housekeeping genes, as used herein, relates to genes which code
for proteins which are constitutively expressed and carry out essential cellular functions.
Preferred housekeeping genes for use in the present invention include β-2-microglobulin,
ubiquitin, 18-S ribosomal protein, cyclophilin, GAPDH and actin.
[0096] In another embodiment, the invention relates to the use of a kit of the invention
for predicting the clinical outcome of a subject suffering from gliobastoma multiforme,
wherein if said agents detect a high expression levels and/or high levels of polysomy
/ amplification of EGFR gene and low expression levels and/or high LOH levels of PTEN
gene, with respect to y standard reference values, then the clinical outcome of the
subject is high.
[0097] Methods for detecting the expression levels of EGFR and PTEN and the methods for
determining the polysomy of EGFR and the LOH of PTEN as well as the standard reference
values has been described previously.
[0098] In another embodiment, the invention relates to the use of EGFR expression levels
and/or polysomy / amplification levels of EGFR gene and the expression levels and/or
LOH levels of PTEN gene as predictive marker of the outcome of a glioma patient.
[0099] In a preferred embodiment, the glioma is a gliobastoma multiforme (GMB).
[0100] In another embodiment, the invention relates to the use of erlotinib and/or temozolomide
in the manufacture of a medicament for the treatment of a glioma, wherein the medicament
is for subjects having a low expression levels and/or high LOH levels of PTEN gene
with respect to standard reference values and high expression levels and/or high polysomy
/ amplification of EGFR gene with respect to said standard reference values.
[0101] In a preferred embodiment, the glioma is a gliobastoma multiforme (GMB).
EXAMPLES
I. MATERIAL AND METHODS
Patients and specimens.
[0102] Multiple formalin-fixed, paraffin-embedded blocks from fifty-six (56) primary brain
tumor specimens were obtained from the Hospital Universitari de Belvitge. These patients
were part of a longitudinal cohort and selected utilizing pre-determined inclusion
criteria which included a primary diagnosis of any one of the following criteria:
astrocytic, oligodendrocytic and glial tumor, with continuous follow-up for a median
of 3 years and available tissue material. Clinical data was abstracted from the patient's
clinical records utilizing a set of pre-determined clinical-pathologic and outcome
data fields (see Table 1). The histologic breakdown of the tumor specimens were as
follows: 1. Astrocytoma group, total 44 patients: (1) pilocytic, (13) diffuse, (16)
anaplastic, and (14) glioblastoma multiforme; II. Oligodendroglioma group, total 6
patients: (6) oligodendroglioma; and
III. Mixed, total four patient: (3) oligoastrocytoma, and one (1) anaplastic oligoastrocytoma.
A comprehensive review of the available clinical data files was performed on all patients
in which formalin-fixed, paraffin-embedded (FFPE) samples / blocks were available
for further analysis. 5 µm sections were obtained from all blocks, stained with hematoxylin
and eosin (H&E) and reviewed by two pathologists to assess overall tumor content =
50% tumor in at least one 200X field), necrosis and overall quality (e.g. thermal
cautery effect, appropriate fixation with acceptable morphology etc). Subsequent sections
were then obtained for DNA FISH assays and quantitative immunofluorescence as outlined
below. All blocks were retained for subsequent studies including RT-PCR.
Table 1. Clinical data fields for entire cohort with breakdown by diagnostic group |
|
Glioblastoma |
Astrocytoma |
mixed/ others |
Non available |
N=15 |
N=26 |
N=5 |
N=10 |
Patients gender: |
Male |
8 |
17 |
4 |
5 |
|
Female |
7 |
9 |
1 |
3 |
|
Non available |
0 |
0 |
0 |
2 |
Patients race |
caucasian |
15 |
25 |
5 |
8 |
|
Non available |
0 |
1 |
0 |
2 |
Symptoms seizures |
Yes |
4 |
13 |
2 |
5 |
|
No |
11 |
13 |
2 |
3 |
|
Non available |
0 |
0 |
1 |
2 |
Sypmptoms high intracranial |
yes |
7 |
5 |
1 |
1 |
pressure |
No |
8 |
21 |
3 |
7 |
|
Non available |
0 |
0 |
1 |
2 |
Number of lesions in |
image 1 |
12 |
21 |
5 |
7 |
pretreatment |
Image 2 |
1 |
1 |
0 |
0 |
|
multiples |
2 |
3 |
0 |
1 |
|
Non available |
0 |
1 |
0 |
2 |
1 location of lesions in |
frontal lobe |
10 |
10 |
3 |
5 |
pretreatment image: |
temoral lobe |
3 |
3 |
0 |
1 |
|
parietal lobe |
1 |
6 |
1 |
0 |
|
operculum |
0 |
1 |
0 |
0 |
|
brain stem |
1 |
2 |
1 |
0 |
|
spinal cord |
0 |
1 |
0 |
0 |
|
optic nerve |
0 |
0 |
0 |
1 |
|
interhemispheri |
0 |
1 |
0 |
0 |
|
c cisure |
|
|
|
|
|
cerebellum |
0 |
0 |
0 |
1 |
|
|
0 |
1 |
0 |
0 |
|
Non available |
0 |
1 |
0 |
2 |
2 location of lesions in |
temoral lobe |
1 |
1 |
0 |
1 |
pretreatment image |
parietal lobe |
3 |
1 |
0 |
0 |
|
occipital lobe |
2 |
1 |
0 |
1 |
|
corpus callosum |
0 |
1 |
0 |
0 |
|
basal ganglia |
2 |
0 |
0 |
0 |
|
brain stem |
0 |
1 |
0 |
0 |
|
thalamus |
0 |
0 |
0 |
1 |
|
Non available |
7 |
21 |
5 |
7 |
1 side of lesions in pretreatment |
Right |
9 |
12 |
2 |
6 |
image |
Left |
4 |
11 |
3 |
2 |
|
Unknown |
0 |
1 |
0 |
0 |
|
Non available |
2 |
2 |
0 |
2 |
2 side of lesions in pretreatment |
Left |
1 |
1 |
0 |
1 |
image |
Non available |
14 |
25 |
5 |
9 |
Type of specimen |
biopsy |
1 |
6 |
0 |
4 |
|
stereotaxic |
2 |
4 |
0 |
1 |
|
biopsy |
|
|
|
|
|
Resection |
12 |
16 |
5 |
3 |
|
Non available |
0 |
0 |
0 |
2 |
Type of postsurgical removal |
complete |
4 |
11 |
1 |
6 |
|
Incomplete |
10 |
12 |
4 |
2 |
|
Non available |
1 |
3 |
0 |
2 |
Radiation therapy |
yes |
7 |
12 |
3 |
1 |
|
No |
8 |
12 |
2 |
7 |
|
Non available |
0 |
2 |
0 |
2 |
Type of radiation |
focal |
5 |
8 |
0 |
1 |
|
Non available |
10 |
18 |
5 |
9 |
Total dose of radiation (* *) (Available data: n=20) |
|
60.0 (60.0-60.0) |
60.0 (40.0-60.0) |
51.0 (48.0-54.0) |
60.0 (60.060.0) |
Chemotherapy |
Yes |
2 |
9 |
1 |
1 |
|
No |
13 |
15 |
4 |
7 |
|
Non available |
0 |
2 |
0 |
2 |
Type of first chemotherapy |
BCNU |
0 |
7 |
0 |
0 |
|
TMZ |
2 |
1 |
0 |
0 |
|
PCV |
0 |
1 |
0 |
1 |
|
Non available |
13 |
17 |
5 |
9 |
Recurrence or progression |
Yes |
4 |
13 |
4 |
6 |
|
No |
9 |
10 |
1 |
1 |
|
Non available |
2 |
3 |
0 |
3 |
Type of first recurrence |
focal |
3 |
8 |
4 |
6 |
|
Diffuse |
1 |
1 |
0 |
0 |
|
Multiple |
0 |
2 |
0 |
0 |
|
Non available |
11 |
15 |
1 |
4 |
Treatment of first recurrence |
Yes |
3 |
10 |
4 |
5 |
|
No |
1 |
1 |
0 |
1 |
|
Non available |
11 |
15 |
1 |
4 |
1 type of treatment of first |
Radiation |
0 |
2 |
1 |
1 |
recurrence |
Chemotherapy |
2 |
3 |
1 |
0 |
|
Surgery |
1 |
4 |
2 |
4 |
|
Radiosurgery |
0 |
1 |
0 |
0 |
|
Non available |
12 |
16 |
1 |
5 |
2 type of treatment of first |
Radiation |
0 |
2 |
0 |
4 |
recurrence |
Chemotherapy |
0 |
1 |
0 |
0 |
|
Non available |
15 |
23 |
5 |
6 |
3 type of treatment of first |
Chemotherapy |
0 |
1 |
0 |
1 |
recurrence |
Non available |
15 |
25 |
5 |
9 |
Kamovsky value pretreatment |
|
70.0 (40.0- |
90.0 (20.0- |
95.0 |
100 (60.0- |
(**) (Available data: n=49) |
|
100) |
100) |
(90.0-100) |
100) |
Kamovsky value postreatment |
|
60.0 (30.0- |
80.0 (10.0- |
95.0 |
100 (30.0- |
(**) (Available data: n=46) |
|
90.0) |
100) |
(80.0-100) |
100) |
Death of patient |
yes |
15 |
15 |
1 |
1 |
|
No |
0 |
7 |
3 |
5 |
|
Non available |
0 |
4 |
1 |
4 |
DNA EGFR and PTEN FISH
[0103] Using the LSI EGFR/CEP 7 dual color probe (Vysis, Abbot Molecular) for EGFR and the
LSI PTEN (10q23) / CEP 10 dual color probe for PTEN (Vysis, Abbot Molecular), DNA
FISH including hybridization, washing and fluorescence detection was performed on
all specimens in the cohort as per standard protocols (
Smith et al., 2001. JNCI; 93:1246-1256). Briefly, paraffin sections were dewaxed in xylenes, microwaved in 10mmol/L sodium
citrate (pH 6.0) solution for 5-10 minutes, cooled to room temperature, rinsed, and
then treated with pepsin HCL for 5 minutes at 37C before being rinsed and dehydrated.
The prewarmed probe mixture was applied to the slides, and a cover-slip sealed in
place with rubber cement. The slides were then denatured at 85°C for 4 to 6 minutes
using an automated hybridization chamber and then incubated overnight at 37C. After
standard post hybridization washes the slides were stained with the DNA stain DAPI
and mounted with antifade (Vectashield). Prior to imaging all H&E slides were evaluated
by a pathologist and regions of interest were identified based on histopathologic
and quality criteria including tumor content, appropriate fixation, necrosis and vascularity.
A H&E image of individual GBM cases is illustrated in Figure 5A. All slides were imaged
using a Nikon immunofluorescent microscope by a trained scientist blinded to outcome.
Criteria for FISH anomalies were defined by use of histologically normal brain specimens.
Simple gain required 10% or more of nuclei with three or more locus-specific probe
signals. Loss of the q arm of chromosome 10 required the overall mean PTEN / CEP10
ratio to be less than 0.90. Amplification was applied for both EGFR and PTEN and required
that the ratio must be 2 or more and that 10% or more of nuclei had more than three
EGFR and or PTEN signals. Representative regions of interest were acquired for documentation
of signal in all specimens (Examples of representative images of FISH AMP/HP of EGFR
and monosomy of PTEN are shown in figure 5B and C respectively). For EGFR FISH we
followed the
Cappuzzo F, et al. (JNCI 2005;4: 643-55) classification scheme and identified 6 categories for EGFR status in all tumor specimens.
These individual states included: D=disomy, HT=high trisomy, LT=low trisomy, HP=high
polysomy, LP=low polysomy, and AMP=amplification. In this schema both HP and AMP together
are considered amplification. PTEN status was characterized as D=disomy, LOH=>10%,
P=polysomy, AMP=amplified and NE=not evaluable. All cases were reviewed by a single
observer and recorded anonymously with respect to tumor type or outcome.
Quantitative Immunofluorescence (IF)
[0104] Utilizing previous established methods (
Cordon Cardo et al., 2007 JCI 2007;117;1876-83;
Donovan et al., 2008 JCO;26:3923-3929) the multiplex- 1 (mplex1) assay was constructed incorporating the following antibodies:
GFAP (glial fibrillary acidic protein), PTEN (phosphatase and tensin homolog), PAKT
and Ki67 combined with the nuclear stain DAPI (4',6-diamidino-2-phenylindole) - see
table 2 for a complete review of antibodies used within the multiplex1 assay. The
individual antibodies were evaluated in simplex IF assays using control tissues and
cell lines to confirm expression sensitivity and specificity prior to inclusion in
the multiplex assay.
Table 2. Reagent list.
Antibody |
Vendor |
Catalog# |
Dilution |
lsotype |
Labels |
samples |
|
|
|
1:30 |
MIgG1 |
2X M 488 |
GBM cases, NL brain, LNCaP, |
PTEN |
Neomarkers |
|
|
|
|
Tonsil |
Ki67 |
Dako |
|
1:100 |
MIgG1 |
2X M 555 |
same |
pAKT |
CST |
#3787 |
1:25 |
RIgG |
2X R 594 |
same |
GFAP |
Dako |
|
1-200 |
MIgG1 |
2X M 647 |
same |
[0105] In brief, FFPE samples were de-paraffinized, rehydrated and subjected to an antigen
retrieval process with the Reveal buffer system (Biocare Medical). A series of four
antibodies were combined with DAPI into a multiplex immunofluorescent assay. The reagents
listed in Table 2 were differentially labeled and with the identified fluorochromea
and then sequentially applied onto individual sections using a Nemesis 7200 immunostainer
(BioCare Medical). A minimum of three images or regions of interest (ROI) were acquired
by a pathologist (blinded to outcome) using a Nikon 90i immunofluorescent microscope
equipped with a CRI spectral imaging system (CRI). Utilizing pre-developed unmixing
libraries and CRI software, individual gray scale images were generated which represent
the antibody - filter combination under investigation. The individual images are evaluated
for signal:noise ratio, cellular distribution and co-distribution with other antibody-fluorochrome
labeled reagents. Using the existing CRI software manual thresholds were created for
individual antibody-fluorochrome combinations to maximize signal:noise and preserving
distribution. Quantitative metrics were generated using the software to identify percentages
of individual cell populations exhibiting a positive signal (based on the threshold),
normalized to the tumor region under evaluation. A series of prostate cancer cell
lines including LNCaP, PC3 and DU145 (ATCC) were obtained, grown to confluency, harvested
as agar cell pellets, fixed and embedded in paraffin and then all three cell lines
were placed into a cell array for quality control both during assay development and
investigation of brain tumor cases.
Results and Discussion
Patients and specimens
[0106] There were 54 patients with available clinical records and FFPE tissue samples appropriate
for evaluation. The breakdown of patients is listed in Table 3 with the majority of
patients in the astrocytoma diagnostic category. The individual demographic features
including sex, location of mass, treatment and outcome data are outlined in detail
in Table 1.
Table 3. Diagnosis category of the patients.
Original codes |
Pure GBM |
Astrocytoma |
Oligodendroglioma |
Mixed/ other |
001: astrocytoma grade II |
0 |
10 |
0 |
0 |
002: astrocytoma grade III |
0 |
16 |
0 |
0 |
003: glioblastoma |
15 |
0 |
0 |
0 |
004: oligoastroctoma grade II |
0 |
0 |
0 |
3 |
005: oligoastrocytoma grade III |
0 |
0 |
0 |
2 |
006: oligodendroglioma grade II |
0 |
0 |
4 |
0 |
007: oligodendroglioma gradeIII |
0 |
0 |
1 |
0 |
008: pilocytic, astrocytoma |
0 |
2 |
0 |
0 |
009: gliomatosis cerebri 999: Non available |
0 |
0 |
0 |
1 |
999 : Not available |
0 |
0 |
0 |
2 |
[0107] In order to understand general survival trends within the cohort and in particular
the glioblastoma group vs. others we performed Kaplan-Meier survival function analyses
to estimate survival between the three groups. Figure 1 is the Kaplan-Meier survival
curve demonstrating reduced survival in the glioblastoma group vs. the anaplastic
astrocytoma and mixed categories (log-rank test P=0.001). Figure 2 is a second Kaplan-Meier
survival function curve which evaluates a more composite end-point which includes
progression free and overall survival in the same three diagnostic categories (log-rank
test P<0.001). In agreement with the literature, the glioblastoma group has a reduced
overall and progression free survival when compared to the astrocytoma and pure oligodendroglioma
group which has the longest survival time of all three diagnostic categories. As evident
from the curves, a subset of the anaplastic astrocytoma group appears to behave like
the glioblastoma category.
EGFR and PTEN FISH
[0108] Given the frequency of EGFR amplification (approximately 40%) and reported role that
EGFR plays in the development of glioblastoma, the first DNA FISH study performed
was on the characterization of EGFR. Using the scoring methods of both
Cappuzzo et al., 2005 JNCI;4:643-55and
Smith et al., 2001 JNCI;93:1246-1256, we investigated EGFR profiles in all brain tumor cases in the group. Given the overall
cohort size we opted to further classify patients as positive if they exhibited either
polysomy and/or amplification of the EGFR locus or chromosome 7. This is in contrast
to some reported studies which independently characterize EGFR DNA FISH as either
polysomy or amplification. In our study 71% of the GBM patients had EGFR polysomy
/ amplification compared with 45% in the anaplastic astrocytoma group. We did not
find EGFR amplification or polysomy to be associated with survival when examining
the entire cohort or individual subgroups (all patients log-rank test p=0.8; glioblastoma
only patients p=0.1). Dual-color FISH was also performed with probes for PTEN utilizing
a similar scoring system as EGFR; however in addition to disomy and polysomy we included
the loss of PTEN as 10% in tumor epithelial nuclei. In our studies 61% of GBM samples
had PTEN LOH compared with 25% in the anaplastic astrocytoma group. There is sparse
literature on the evaluation of PTEN by DNA FISH in brain tumor samples with most
studies using more molecular techniques such as sequencing, RT-PCR and immunohistochemistry.
Noteworthy in our cohort, PTEN LOH was univariately and statistically associated with
reduced survival in the combined glioblastoma, astrocytoma and mixed patient group
(n=25 without LOH, median survival 913D vs. n=10 with LOH, median survival 174D; log-rank
test p=0.04) See figure 3.
[0109] Neither EGFR polysomy / amplification or PTEN loss were significant, independent
predictors for survival in the glioblastoma only group (p=0.1); however, when combined
we observed a trend towards significance with an increase in overall survival (n=6
with both EGFR/PTEN, median survival 242D vs. n=6 without, median survival 71D; log-rank
test p=0.034) (see Figure 4) The hypothesis is that the combination of PTEN LOH and
EGFR amplification in glioblastoma may represent a subset of patients with a tumor
phenotype more amenable to radiation and/ or temozolomide treatment.
Tables 4.Clinical characteristics of patients with previous filter conditions.
Idalthia |
gender |
age |
sympseiz |
sympticp. |
imagelesions |
imagelocat_1 |
imagelocat_2 |
imageside_1 |
imageside_2 |
diagn |
L08-00080 |
female |
50 |
no |
yes |
1 |
frontal lobe |
NA |
left |
NA, |
glioblastoma |
L06-00084 |
female |
65 |
no |
yes |
1 |
frontal lobe |
NA |
right |
NA. |
glioblastoma |
L08-00035 |
male |
71 |
no |
yes |
1 |
temporal lobe |
NA |
right |
NA |
glioblastoma |
L08-00087 |
male. |
66 |
no |
no |
1 |
frontal lobe |
parietal lobe |
right |
NA |
glioblastoma |
L08-00090 |
female |
74 |
no |
no |
1 |
parietal lobe |
occipital lobe |
right |
NA |
glioblastoma |
L08-00091 |
male |
68 |
no |
no |
1 |
temporal lobe |
NA |
right |
NA |
glioblastoma |
L08-00094 |
female |
66 |
yes |
no |
1 |
frontal lobe |
temporal lobe |
right |
NA |
glioblastoma |
L08-00097 |
male |
50 |
yes |
yes |
1 |
temporal lobe |
NA |
right |
NA |
glioblastoma |
L08-00098 |
female |
49 |
yes |
no |
1 |
frontal lobe |
parietal lobe |
left |
NA |
glioblastoma |
L08-00106, |
male |
66 |
yes |
yes |
multiples |
frontal lobe |
basal ganglia |
NA |
NA |
glioblastoma |
L08-00109 |
female |
60 |
no |
no |
2 |
frontal lobe |
occipital lobe |
left |
NA |
glioblastoma |
L08-00128 |
male |
37 |
no |
yes |
1 |
frontal lobe |
NA |
right |
left |
glioblastoma |
table4.
Idalthia |
diagnspec |
psiremoval |
recur progr |
recurtype |
death |
L09-00080 |
resection |
complete |
yes |
NA |
yes |
L08-00084 |
resection |
compete |
yes |
focal |
yes |
L08-00085 |
resection |
complete |
yes |
focal |
yes |
L08-00087 |
resection |
incomplete |
no |
NA |
yes |
L08-00090 |
biopsy |
incomplete |
no |
NA |
yes |
L09-00091 |
resection |
complete |
no |
NA |
yes |
L08-00084 |
resection |
NA |
no |
NA |
yes |
L08-00097 |
resection |
incomplete |
no |
NA |
yes |
L08-00098 |
resection |
incomplete |
yes |
focal |
yes |
L08-00106 |
stereothaxic biopsy |
incomplete |
no |
NA |
yes |
L08-00108 |
stereothaxic biopsy |
incomplete |
no |
NA |
yes |
L08-00128 |
resection |
incomplete |
yes |
diffuse |
yes |
[0110] VARIABLE INFO Table 4:
gender = "patients gender"
age = "patients age"
sympseiz = "symptoms seizures"
sympticp = "sypmptoms high intracranial pressure"
imagelesions = "number of lesions in pretreatment image"
imagelocat_1 = "location of 1st. lesion in pretreatment image"
imagelocat_2 = "location of 2nd. lesion in pretreatment image"
imageside_1 = "side of 1st. lesion in pretreatment image"
imageside_2 = "side of 2nd. lesion in pretreatment image"
diagn = "pathological diagnosis"
diagnspec = "type of specimen"
psiremoval = "type of postsurgical removal"
recur.progr = "recurrence or progression"
recurrtype = "type of first recurrence"
death = "death of patient"